Blood tests provide useful information but generally only indicate the current status of a patient. Many clinicians have wished for a way to determine the physical condition of a patient in retrospect over a period of time. In a number of diseases such as diabetes, it would be useful to determine the recent changes in health status in a patient and correlate such changes with the patient's own recollection of diet, exercise and medication. However, currently, there are few easily assayed records of physiological changes.
Previous infections have been detectable by the presence of antibodies. However, measuring antibodies is indicative of only a few conditions and generally does not indicate how long it has been since exposure to the antigen unless it is recent enough for IgM antibodies to be present. Metabolic changes in the past are generally not detectable using currently commercially available assays.
During the average 120 days of red blood cell (RBC) life, the RBC are exposed to the entire body and may undergo many changes in response to the body condition.
The most prominent red blood cell component is hemoglobin. Hemoglobin is a molecule which contains a protein portion (globin) and an organometallic portion (heme). This molecule is responsible for delivering oxygen throughout the body. A number of compounds modify hemoglobin, some of which affect its activity. For example, carbon monoxide binds to hemoglobin, making it unable to carry oxygen and thus poisons the molecule. Certain drugs react with hemoglobin to form sulfhemoglobin.
Hemoglobin also may be glycosylated. The resulting molecule, glycohemoglobin, retains its original ability to carry oxygen. There are at least five different forms of glycosylated hemoglobin. For example, hemoglobin may be glycated with glucose, galactose, xylose, other non-metabolized sugars, or with phosphorylated sugars such as ribose-5-phosphate or 5-deoxyxylose. The rate at which hemoglobin is glycosylated to form glycohemoglobin is dependent on the sugar concentration (for example, glucose) in the circulating plasma around the red blood cells at the time of exposure, or on the concentration of sugar accumulating within the red blood cell. It has been proposed to use such measurements as a diagnostic test for diabetes, to determine whether a patient is complying with a particular diet or therapy, and to evaluate the effectiveness of a particular therapy. While the glycation of other proteins causes permanent, cumulative damage, hemoglobin is the short term, readily accessible record of such complications.
Several U.S. Patents involve measuring the proportion of glycohemoglobin to hemoglobin such as 4,399,227, 4,647,654, 4,448,888, 4,438,204, 4,372,747, 4,465,774, 4,268,270 and 4,835,097. The '097 patent teaches the use of this ratio as a clock for determining the age of individual red blood cells and correlating this age with the level of blood sugar or of a drug in the circulation. In recent years glycohemoglobin measurements have attracted attention as a possible indicator of diet in diabetic patients. Nakashima et al, Clinical Chemistry, 35(6): 958-62(1989), attempt a correlation with blood sugar at the time the glycohemoglobin sample was taken.
Other chemicals may be measured in red blood cells. Alcohol causes a different sugar molecule to become trapped on the hemoglobin molecule due to the indirect action of alcohol on the normal breakdown of sugar in the red blood cell. The altered hemoglobin contains 5-deoxy-D-xylulose-1-phosphate (DXP) and is called "DXP-hemoglobin." Hoberman, U.S. Pat. No. 4,463,098, has proposed a method for determining the historic record of alcohol consumption or abnormal alcohol in the blood by measuring DXP-hemoglobin. However he does not seek to measure hemoglobin with altered glycosylation from non-alcohol related sources. Hoberman's meaning of "historic record" is of general history averaged over the lifespan of 120 days. He does not seek to provide a detailed, calendarized history of alcohol consumption.
Over time erythrocytes (RBC) slowly change density, and may be separated according to age based on their relative densities, Leif et al, Biochemistry, 51:520-28(1964). As erythrocytes age, they lose water without significant loss of solids and therefore become more dense, Leif et al, Proceedings of the National Academy of Sciences, 51: 520-8(1964). A number of other biochemical changes also occur in erythrocytes over time which has led to various proposals to assay for the age of erythrocytes. See U.S. Pat. No. 4,835,097. While some successes at separating red blood cells have been published, this technique has suffered from a number of problems which have prevented its general clinical use. Previous methods for ordering red blood cells by density, i.e., by age, have been cumbersome. These methods generally involve carefully preparing density gradients and placing the desired sample on top. A centrifugation step follows and harvesting of cells at each layer completes the process. Tests which correlate density layers with age include gradual loss of enzyme activity and also gradual increase in hemoglobin concentration and loss of cell volume.
The present invention has overcome the deficiencies of prior blood cell ordering techniques by variable speed centrifugation of RBCs in plastic-coated glass capillary tubes and improved treatment of red blood cells for deformability and contrast of density.
Varying speed centrifuge techniques such as Wissler, U.S. Pat. No. 4,343,793, have been used to obtain thrombocytes and leukocytes from blood. The blood is centrifuged at low speed whereby all the red blood cells and the leukocytes are sedimented together while the plasma constituents and the thrombocytes remain in the supernatant. The supernatant rich in thrombocytes and plasma constituent is separated from the plasma constituents by brief centrifugation at a higher speed. Wissler did not seek to order red blood cells by age.
Others have used sequential different speeds of centrifuging for various purposes. Rogers, in U.S. Patent No., used several centrifugal forces for harvesting "neocytes" from whole blood for transfusion. However, Rogers only separated two layer, and neither analyzed nor reported the history of a condition.
Von Behrens, in U.S. Pat. No. 3914,985, conducted a low force centrifuge step, followed by a high force centrifugation in a separate container. Von Behrens measured the relative volume of various blood cell types after the second centrifugation. Although he refers to "harvesting", Von Behrens did not harvest cells for measuring a chemical history, nor did he evaluate the history of a condition from either red blood cells or other types of cells.
Nakashima, supra, used a first, low centrifugal force to transfer cells into a capillary, and followed with a second, greater centrifugal force for separating cells into age fractions. Nakashima achieved varying degrees of success in separating fractions by density, but the methods were not optimized for commercial use. Nakashima did not consider the freshness of the samples, and there was no analysis for correlating harvested fractions with density.
McEwen et al., U.S. Pat. No. 4,828,716, disclose a centrifuge assembly for blood separation into serum or plasma and "cellular component" in which the velocity of the linear actuation of the motor is accomplished by a control computer in accordance with sensors which relate the color and degree of turbidity of the separated fluid. The computer uses the signals produced by the optical sensors to determine when optimal separation of the sample has occurred, and stops the motor when the separation process is complete.
Sullivan, in U.S. Pat. No. 4,087,567, discloses an anticoagulant coating suitable for coating the interior surfaces of a blood collection tube, such as a capillary tube. The coating consists essentially of ethylene diamine tetraacetate held in a matrix of polyvinyl pyrrolidone, both of which are dissolved in a water-alcohol mixture to form a coating solution. This is merely to provide an anticoagulant coating for blood collection tubes.
Burns et al., U.S. Pat. No. 4,822,745, disclose a method for determining the reticulocyte population in a blood sample by determining the average cell size of the blood sample, partitioning the sample by centrifugation through a medium of known density so as to provide a fraction enriched with neocytes, determining the average cell size of the fraction, comparing the average cell size of the sample to the average cell size of the fraction and using the comparison to provide a determination of the population of reticulocytes in the sample. This technique, however, is merely for quantifying the reticulocytes in blood, rather than to separate cohorts of red blood cells by age.
Levine, in European patent application 0 536 658 A1, discloses a method for obtaining differential erythrocyte counts. In this case, a blood sample is centrifuged in a transparent tube containing plastic beads which are selected to include groups of beads wherein each group has a different sharply defined specific gravity, and which are distributed within the range of red cell densities. The beads form spaced narrow bands in the erythrocyte layer, which bands form boundaries between the different cell subset layers. The lengths of the different cell subset layers are measured to quantify the red cell subsets in the patient's blood. This method is for obtaining a record of the historical formation and/or loss of red cells in a patient's blood for as much as the previous 120 days. The markers may be plastic beads, latex spheres, or liposomes or the like. Each type of marker has a sharply defined specific gravity which lies within the range of specific gravity for erythrocytes.
European patent application 0 126 390 discloses a fluid transfer device formed to have a cannula for piercing a stopper and surrounding shroud to guide the cannula during piercing.
Glass capillary tubing has been coated previously using a polymer. Springston, U.S. Pat. No. 4,966,785, discloses a method of coating the interior surfaces of glass capillary columns with a stationary polymer phase to minimize progressive phase loss and diminished efficiency of the polymer coating. This coating is used for increasing the chemical adsorption properties, not for cell separation. This is a well known coating which is used here for cross polymerization. These columns are used for gas chromatography. The criterion for the interior coating is stability of the coating to solvents used for washing. There is no disclosure of using these tubes for centrifugation, and no cells are harvested for measurement.
Ohayon, in French patent 2 555 074, discloses separating blood in a plastic or glass tube in which the interior is coated with a film of a copolymer of vinylpyrrolidone and vinyl acetate. The tube contains micronized styrene polymers carrying on their surface hydrated micronized silica. This tube is for separating coagulated blood into a light phase consisting essentially of serum and a heavy phase formed essentially of cellular and fibrillar materials. The tube disclosed by Ohayon aids in coagulating the blood introduced thereinto within a period of about three minutes rather than about 30 minutes. There is no indication that red blood cells can be separated by density-only that the serum can be rapidly separated from the coagulated solid materials in the blood using the combination of the coated tube and the beads of micronized silica.
A number of techniques, including simple decanting have been tried to separate the red blood cells from the remaining components of the blood after centrifugation. Farr, U.S. Pat. No. 3,355,098, discloses an open plunger tube within which is a plastic flexible small bore tube whose lower end extends through an opening in the piston head. After centrifuging, the plunger tube is inserted into the centrifuge tube. As the head of the plunger is pressed into the centrifuge tube, the air above the serum is released to the atmosphere, and serum can be extracted through the capillary tube without disturbing the solids.
Taylor, U.S. Pat. No. 3,705,018, discloses a plug for sealing a test tube which permits access to the tube for removal of some or all of the contents thereof. A U-shaped flow tube extends removably through a pair of spaced-apart apertures in the plug. Withdrawal of one tube end from one of the apertures and application of a negative or positive pressure forces fluid materials into or out of the test tube.
Ayres, U.S. Pat. No. 3,850,174, discloses an assembly for separation of blood into a light liquid phase of serum or plasma and a heavy phase including means for pushing a piston member downwardly in a container. The piston also acts as a closure, and a pointed tubular member can be used to pierce the piston to provide a passage for conducting the separated light liquid phase from one side of the stopper-piston to the other side thereof.
Rogers, U.S. Pat. No. 4,416,778, discloses a method for preparing neocyte-enriched blood wherein a sample of whole blood is centrifuged at high speed to distribute the blood components in the chamber along a density continuum. The container system used contains two chambers. Near the end of the centrifuge cycle, when the container has slowed to a relatively low speed, a valve is opened to permit communication between the two chambers so that the older, dense blood cells distal to the spin axis flow through the conduit into the other chamber. Thus, the second speed in Rogers is for the purposes of transferring cells form one compartment to another.
None of the above-cited patents describes a process for separating red blood cells into refined, multiple fractions of red blood cells which can be sequentially harvested by age and further analyzed to provide a history of a condition.